Proteins are the substances responsible for the modulation of numerous biological processes in living organisms, and understanding their intrinsic properties and the study and modulation of their interactions with other substances has become more important for developing treatments for various diseases. One of the major obstacles to the investigation and utilization of proteins is mass-production of proteins. In particular, a bacterial system useful for mass-production of proteins cannot be applied to many human-derived proteins, and thus animal cells or plant cells are currently used for the production of human-derived proteins despite the low production yield.
Repeat proteins refer to all proteins of specific unit modules. Particularly, LRR (Leucine Rich Repeat) family proteins refer to consisting of an assembly of diverse leucine-rich repeat modules at defined positions. LRR family proteins contain an N-terminal region, a variable number of repeat modules, and a C-terminal region to forma stable structure. The repeat module of LRR family proteins has the following characteristics; (1) it has at least one repeat module; (2) the repeat module consists of 20 to 30 amino acids; (3) the repeat module has a conserved pattern of “LxxLxxLxLxxN” (SEQ ID NO:20 and 21), wherein L represents a hydrophobic amino acid such as alanine, glycine, phenylalanine, tyrosine, leucine, isoleucine, valine, and tryptophane, N represents asparagine, glutamine, serine, cysteine or threonine, and X represents any amino acid. LRR family protein further has the characteristics of varying the number of repeat modules and the shape of the C-terminal region, enlarging the binding area with other proteins, and making a new binding site. Representative examples of LRR family protein include Toll-Like Receptor protein (TLR) involved in human immunity, Variable Lymphocyte Receptor (VLR) involved in hagfish immunity, Ribonuclease Inhibitor protein (RI) or the like. Each of them is utilized in immunological or molecular biological studies, and they are usually commercialized and utilized in the studies by production in insect or animal cells, isolation of VLR from hagfish after injection of antigens, or direct extraction of RI from placenta. One more example is the use of VLR that is produced after refolding of inclusion bodies expressed in E. coli. It is believed that such difficulties in mass-production of soluble LRR family proteins in bacteria such as E. coli may be attributed to obstruction of consecutive hydrophobic residues of LRR family protein in the expression of the soluble form. VLR proteins are involved in the immunity of jawless fish, and are similar to antibodies in constitution and utilization. Thus, if their mass-production is possible, they are expected to possess broad applicability like antibodies. In addition, it is known that LRR family proteins are involved in diverse physiological processes such as signal transduction, cell cycle regulation, apoptosis and immune response. Thus, there is an urgent need to develop a technique capable of mass-producing LRR family proteins in a soluble form in host cells such as E. coli, because the technique would make it possible to extend the applications of LRR family protein in the biological and medical fields.
Meanwhile, a novel therapeutic protein which binds with a specific target, for example MD-2 (Myeloid differentiation protein-2) protein which is used as an agent for sepsis, could be prepared by various methods using LRR family protein. The preparation method for the polypeptide bound with a specific target is effectively accomplished by designing is based on a structure.
Sepsis is a highly fatal disease caused by immune hypersensitivity reactions due to bacterial infection, which is characterized by thrombosis, multiple organ dysfunction syndrome, and high mortality (60%). The most common cause of sepsis is lipopolysaccharide (LPS) which is the major component of the outer membrane of Gram-negative bacteria, and the immune responses are known to be initiated by recognition of bacterial LPS infected in blood by immune cells.
TLRs (toll-like receptors), found on the surface of human immune cells, play a major role in the induction of innate immunity, and LPS initiates signal transduction through TLR4 among TLRs, which results in the release of cytokines and the induction of immune responses. This immune response is a normal event in the body, but progression of immune hypersensitivity reactions in people whose immune system is weakened by disease or extreme stress may lead to severe sepsis. However, LPS does not directly bind to the TLR4 receptor which is one of the innate immune receptors, and LPS binds to a TLR4/MD-2 complex by mediation of MD-2 (Myeloid differentiation protein-2) to initiate the signal transduction. Therefore, recent studies have been made to develop a therapeutic agent for sepsis, which targets TLR4, MD-2, or TLR4/MD-2 complex to block the LPS signal transduction.
Currently only one drug, Xigris (Eli Lilly), is FDA-approved specifically for sepsis therapy, and is a recombinant version of naturally occurring APC (activated protein C) that has an anti-thrombotic effect. Xigris is anticipated to save approximately 15% of the lives of patients with the most severe forms of sepsis. In addition, eritoran was developed by Eisai (Japan) for the purpose of treating sepsis, which is similar to LPS in chemical structure, thereby binding to MD-2 competitively, and it was undergoing phase III clinical trials in the end of 2009. Many other therapeutic agents including antibodies, developed by numerous pharmaceutical companies, have undergone clinical trials. Unfortunately, most of them show no effect or very low effects, thereby bringing no positive outcomes (Triantafilou et al., Expert Rev. Mol. Med. 24; 6(4):1-18, 2004).
Meanwhile, each of the proteins involved in LPS signal transduction, LBP, CD14, MD-2, and soluble TLR4 (sTLR4), are known to inhibit the LPS signal transduction, and thus they are used for the development of a promising therapeutic agent for sepsis. The sTLR4/MD-2 complex is known to show very excellent inhibitory effect on the LPS signal transduction. MD-2 singly forms a non-functional complex, but binding of MD-2 with sTLR4 forms a stable structure, resulting in competition for LPS binding between sTLR4/MD-2 complex and TLR4/MD-2 complex present on the surface of immune cells (Mitsuzawa et al., J. Immunolo., 177:8133-8139, 2006; Brandl et al., J. Endotoxin Res., 11:197-206, 2005). In addition, each binding affinity (Kd) of CD14, MD-2, and TLR4/MD-2 complex for LPS is reported to be 30 nM, 65 nM, and 3 nM, respectively, and the higher inhibitory effect of sTLR4/MD-2 complex on LPS than CD14 and MD-2 is attributed to such difference in the binding affinity (Akashi et al., J. Exp. Med., 198(7):1035-1042, 2003).
However, there is a drawback in that the high molecular weight and insoluble expression of cell membrane protein make it difficult to produce sTLR4. In order to solve this problem, treatment of experimental animals (mouse) with a TV3 protein alone (without MD-2), which was prepared by fusion of a hagfish variable lymphocyte receptor (VLR) with the MD-2 binding site of TLR4, showed inhibitory effects on sepsis (PLoS ONE, 4:e7403, 2009). The TV3 fusion protein is a protein therapeutic agent that is prepared by a hybrid LRR technique of assembling LRR (leucine rich repeat) family proteins such as TLR4, and the hybrid LRR technique is a promising method in development of novel protein scaffolds as alternatives to antibodies as well as in the development of therapeutic agents for sepsis.
Meanwhile, sepsis results from excessive immune response initiated by signal transduction of the innate immune system. Thus, therapeutic agents developed for the treatment of sepsis are required to have a rapid, immune cell-based assay system. That is, protein drugs showing inhibitory effects on sepsis are required to have a cell-based assay system for LPS-induced immune response. Therefore, the present invention intended to develop a cell-based assay system for monitoring the LPS blocking activity of the LRR protein drugs by introduction of an NF-κB dependant firefly luciferase gene into the HEK293 cell line. The present invention also intended to develop a cell-based assay system for monitoring the LPS blocking activity of the protein drugs by analyzing cytokines secreted from the LPS-treated, macrophage-differentiated THP-1. However, there is a problem in that both of the above cell lines showed no LPS blocking activity when sTLR4, TV3, or a protein drug prepared by the hybrid LRR technique is used alone (without MD-2), even though they express TLR4 and MD-2 required for LPS signal transduction.